One of the first hypotheses on the existence of DNA repair mechanisms proposed that the DNA present in the germinal cells of multicellular organisms would be protected against damage, and consequently against aging, by virtue of an efficient DNA repair associated to meiosis. On the other hand, somatic cells would be much more vulnerable to DNA damage as they possess a lesser DNA repair capacity. However, it is now known that there are several DNA repair machineries which also act at a somatic level that eliminate most of the damage and alterations occurring at the genome, and that excessive damage or dysfunction of these protective mechanisms could lead to premature aging and cell death, or else favour the proliferation that characterizes tumoral processes.
In mammals there is a clear correlation between the longevity of different species and their capacity to carry out DNA repair. Furthermore, aging can be accelerated by treatments that damage DNA or due to genetic defects at genes involved in DNA repair. In humans, one of the keys in aging is the degeneration of the central nervous system (CNS) and this is related to its extreme sensitivity to genotoxic agents. It is widely accepted that most degenerative diseases, that sometimes show symptoms of premature aging of the CNS, are due to accumulation of DNA damage.
Moreover, fixation of mutations due to DNA repair defects (somatic mutations) can cause cellular transformation. Carcinogenesis is a complex process initiated by a damage in the DNA, followed by the mutation or translocation of a DNA segment, and ending with a phenotypic transformation of the cell. It is widely documented that some normal genes (proto-oncogenes) can become tumoral (oncogenes) by the action of several agents that produce DNA strand breaks. Frequently, these changes directly affect the sequence of the proto-oncogene, that is translocated to another breaking point in a different chromosome. As known in the art, most cells are endowed with DNA repair mechanisms that recognize and eliminate this genomic damage. The absence or dysfunction of these systems would increase the probability of cell transformation. Poly ADP-ribose polymerase (PARP) was one of the first enzymes to be involved in DNA repair of DNA strand breaks. Its activity was shown to be higher in the nuclei of fibroblast transformed by SV40 than in the controls. Similarly, the enzymatic activity of PARP was shown to be higher in leukemic than in normal cells, and the same increase was observed in the mucosa from colorectal cancer patients than in that derived from healthy individuals [Miwa et al., Arch. Biochem. Biophys. 181:313-321 (1977); Burzio et al., Proc. Soc. Exp. Biol. Med. 149:933-938 (1975); Hirai et al., Cancer Res. 43:3441-3446 (1983)]. This work allows one to conclude that the DNA polymerase activity of PARP is increased after DNA damage. Moreover, inhibition of the polymerization activity of PARP by addition of specific drugs produced an increase in genomic DNA damage and in the risk of oncogenic transformation [Harris, Int. J. Radiat. Biol. 48:675-690 (1985)]. These data led to the suggestion to use PARP as a tumoral marker and also as an indicator of the predisposition to develop cancer, see U.S. Pat. No. 5,449,605.
On the other hand, it has been recently demonstrated that the non-hereditary colorectal carcinomas, and about 15% of sporadic gastric tumors, are characterized by the instability of repetitive sequences in the DNA (microsatellites). This phenotype, named mutator phenotype, is accompanied by a several hundred fold increase in spontaneous mutations [Eshleman et al, (1995), Oncogene 10, 33-37; Eshleman et al, (1996), Oncogene 12, 1425-1432], that appears to be due to dysfunction of genes involved in the process of DNA mismatch repair [Hoffman & Cazaux, (1998), Int. J. Oncol. 12, 377-382; Umar & Kunkel, (1996), Eur. J. Biochem. 238, 297-307]. Thus, the basis for a predisposition to colon cancer can based on the existence of a germ line mutation (inherited) in some of those genes (hMSH2, hMLH1, PMS1, PMS2, hMSH3 and hMSH6) required for postreplicative mismatch repair [Prolla et al, (1998), Nature Genetics 18, 276-279]; a second sporadic mutation, that could appear as a consequence of a defective DNA repair, could increase the mutator phenotype if targets the other allele of the same gene, or targets another gene involved in DNA repair (second mutator), allowing the further selection of proliferative variants, and leading to tumour formation. Several mutations have been described in two human genes involved in mismatch repair (hMSH3 and hMSH6), in patients affected by hereditary colon carcinoma, that frequently consist in frameshifts occurring at series of consecutive adenine and cytosine residues [Yamamoto et al, (1998), Cancer Res. 58, 997-1003].
To date, there is no evidence that Pol β participates in the process of “mismatch repair”. However, a specific deletion (amino acids 208 to 236), close to the active site, in one of the alleles of Pol β has been found associated to some breast and colorectal carcinomas [Battacharyya & Banerjee, (1997), P.N.A.S. USA 94, 10324-10329]. Furthermore, 83% of the human colon carcinomas show the presence of mutations in the Pol β gene [Wang et al, (1992), Cancer Res. 52, 4824-4827; Dobashi et al, (1995), Hum. Genet. 95, 389-390]. There is also reported evidence on the existence of Pol β mutations associated to bladder carcinoma, but in this case there are also additional mutations associated to tumour suppressor genes as p16 and RB [Matsuzaki et al, (1996), Mol. Carcinog. 15, 38-43]. The attempts to demonstrate the importance of Pol β in tumourogenesis were not successful, since germline deletion of Pol β resulted in a letal phenotype [Gu et al, (1994), Science 265, 103-106].
To date, 10 eukaryotic cellular DNA polymerases have been described (DNA polymerases α, β, γ, δ, ε, ζ, η, θ, ι, and κ). Of these DNA polymerases α, β and ε are involved in DNA replication [Wood & Shivji, (1997), Carcinogenesis 18, 605-610]. Most DNA repair mechanisms have associated DNA synthesis steps to replace the damaged nucleotides, or to bridge the ends of broken DNA. One of the mechanisms frequently used in the cell is “base excision repair” which eliminates slightly modified bases or abasic nucleotides. The DNA synthesizing enzyme involved in this process appears to be Pol β, acting in concert with XRCC1 and DNA ligase III [Dianov & Lindahl, (1994), Nature 362, 709-715; Sobol et al, (1996), Nature 379, 183-186; Nicholl et al, (1997), Biochemistry 36, 7557-7566]. There has been speculation about the existence of an alternative, larger scale process, involving the processivity factor PCNA, and DNA polymerases δ and ε. The elimination of thymidine dimers and bulk adducts in the DNA, carried out in a process named “nucleotide excision repair”, appears to involve also DNA polymerases δ or ε. In the same sense, DNA polymerases δ or ε are invoked in catalysing the post-replicative repair of insertion errors (mismatch repair), since this process is normal in Pol β-deficient cells. The expression of Pol β, a house keeping gene, is constant, and is neither stimulated by cell growth nor cell cycle controlled [Zmudzka et al, (1988), Nucleic Acids Res. 16, 9587-9596]. Clearly, since the mismatch repair must be coupled to DNA replication, both processes should be co-regulated. DNA polymerases ζ, η and θ are not appropriate to correct replication errors. On the contrary, their biochemical properties support that these enzymes are involved in the bypass of DNA lessions, an alternative to DNA repair [Lawrence & Hinkle, (1996), Cancer Surv. 28, 21-31; Masutani et al, (1999), Nature 399, 700-704; Sharief et al, (1999), Genomics 1, 90-96].
It therefore remains a problem in the art to identify further DNA polymerases involved in the acquisition of a mutator phenotype. These polymerases may be clinically extremely important for providing the tools for identifying individuals with a predisposition of developing cancer, improve the efficiency of clinic surveillance for an early detection and intervention at early stages [de la Chapelle & Peltomaki, (1995), Annual Rev. Genet. 29, 329-348], and develop novel strategies of therapy based on these targets. It is also possible that some of these DNA polymerases could be involved in neurodegenerative diseases of the central nervous system, since these processes are probably related with a defective or error-prone DNA repair.